*Agnieszka Bogusławska-Kapała1, 2, Agnieszka Piekarska3, Jolanta Ochocińska1, Alicja Cackowska-Lass1, Agata Żółtowska1, Barbara Kochańska1
An evaluation of salivary levels of lysozyme and lactoferrin in patients in the late period after allogeneic hematopoietic stem cell transplantation (HSCT)
Ocena stężenia lizozymu i laktoferyny w ślinie pacjentów będących w późnym okresie po allogenicznej transplantacji komórek hematopoezy (HCT)
1Department of Conservative Dentistry, Medical University of Gdańsk
Head of Department: Associate Professor Barbara Kochańska, MD, PhD
2Department of Integrated Dentistry, Department of Conservative Dentistry, Medical University of Warsaw
Head of Department: Izabela Strużycka, MD, PhD
3Department of Hematology and Transplantation, Medical University of Gdańsk
Head of Department: Professor Andrzej Hellman, MD, PhD
Streszczenie
Wstęp. Transplantacja komórek hematopoezy (ang. hematopoietic cell transplantation – HCT) często łączy się z występowaniem powikłań w jamie ustnej, niejednokrotnie zagrażających zdrowiu i życiu pacjentów. Zaburzenia jakościowe i ilościowe czynników nieswoistej odporności zawartych w ślinie, takich jak lizozym i laktoferyna, mogą przyczyniać się do rozwoju tych powikłań.
Cel pracy. Analiza stężenia lizozymu i laktoferyny w ślinie pacjentów będących w późnym okresie (powyżej setnej doby) po allogenicznej transplantacji komórek krwiotwórczych, z uwzględnieniem czasu, jaki upłynął od transplantacji oraz szybkości wydzielania śliny.
Materiał i metody. Zbadano 45 osób będących 3,5 miesiąca do 5 lat po HCT. Do oznaczenia stężenia lizozymu i laktoferyny w ślinie spoczynkowej i stymulowanej zastosowano metodę ELISA (ang. enzyme-linked immunosorbent assay).
Wyniki. Średnie stężenie lizozymu i laktoferyny w ślinie nie zmieniało się istotnie w zależności od upływu czasu po przeszczepieniu. Obserwowano znaczne wahania stężenia lizozymu. Nie stwierdzono zależności pomiędzy stężeniem lizozymu a szybkością wydzielania śliny. Średnie stężenie laktoferyny było istotnie statystycznie wyższe w grupie pacjentów po HCT w porównaniu z grupą kontrolną i wzrastało istotnie wraz ze spadkiem szybkości wydzielania śliny spoczynkowej i stymulowanej.
Wnioski. W każdym okresie po HCT należy spodziewać się znaczących wahań stężenia czynników odpornościowych zawartych w ślinie, szczególnie u pacjentów poddawanych immunosupresji, z zaburzeniami wydzielania śliny i/lub z chorobą „przeszczep przeciwko gospodarzowi”. Wysokie stężenie laktoferyny w ślinie może stanowić wskaźnik stanu zapalnego w obrębie tkanek miękkich jamy ustnej.
Summary
Introduction. Hematopoietic Stem Cell Transplantation (HSCT) is often associated with oral complications, which frequently affect the health and even lives of patients. Quantitative and qualitative impairment of salivary immunological factors, such as lactoferrin and lysozyme, can contribute to the development of these complications.
Aim. An analysis of salivary lysozyme and lactoferrin levels in patients in the late period (more than 100 days) after allogeneic stem cell transplantation. The time elapsed since transplantation and salivary flow rate were also taken into consideration.
Material and methods. A total of 45 patients 3.5 months to 5 years after HSCT were evaluated. Enzyme-linked immunosorbent assay (ELISA) was used to determine saliva lactoferrin and lysozyme levels.
Results. No significant relationship was found between mean saliva lysozyme and lactoferrin levels and the time elapsed since HSCT. Significant differences in lysozyme levels were observed. No correlation was found between lysozyme levels and salivary flow rate. Mean lactoferrin levels were statistically significantly higher in post-HSCT patients compared to the control group and increased with a decrease in the stimulated and non-stimulated salivary flow rate.
Conclusions. Significant variations in the levels of immunological salivary factors should be always expected after HSCT, particularly in patients under immunosuppression and/or those with graft-versus-host disease. High levels of salivary lactoferrin can be an indicator of oral inflammation.
Introduction
Hematopoietic stem cell transplantation (HSCT) is an increasingly used therapeutic method in a number of diseases, such as hematopoietic proliferative diseases and congenital metabolic and immune disorders (1).
Patient’s own cells (autologous HSCT) or donor’s cells (allogeneic HSCT) are used for transplantation. The regeneration of hematopoietic system after allo-HSCT is a slow process. Quantitative and qualitative impairment of cellular and humoral immunity persists for a long time in most patients (2). Despite significant therapeutic advances, hematopoietic stem cell transplantation involves the risk of a number of complications, which also affect the oral cavity in most patients (2, 3). The most serious complications include inflammation of the oral mucosa and reduced saliva production (3, 4). Reduced salivary secretion leads to changes in saliva composition, including altered levels of nonspecific and specific immune factors (5-7), which may in turn significantly contribute to the development of pathological processes in the oral cavity (8).
Lysozyme and lactoferrin belong to the most important nonspecific salivary immune factors (8-10). Lysozyme (muramidase) is a cationic protein found in the secretion from the serous cells of the salivary glands, gingival crevicular fluid as well as neutrophils, monocytes and macrophages. The enzyme has a broad spectrum of non-specific action on pathogenic microorganisms. It acts primarily on gram-positive bacteria (Streptococcus mutans, Lactobacillus acidophilus), to a lesser degree on gram-negative bacteria (e.g. Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis) and some fungal species (e.g. Candida spp.) (3, 11). Lysozyme causes microbial cell lysis by hydrolysing the bonds between N-acetylglucosamine and N-acetylmuramic acid in cellular wall peptidoglycan (12). Additionally, it can activate bacterial autolysis as well as inhibit bacterial glucose absorption and acid production (11, 12). It was found that lysozyme can also induce microbial aggregation and inactivate some viruses (11). Muramidase also acts as an anti-inflammatory agent by the inhibition of leukocyte chemotaxis and a direct modulation of complement reaction (10). Additionally, the agent is involved in the production of gamma globulins, it accelerates granulation tissue formation (12) and has anticancer properties (13).
Lactoferrin is a glycoprotein belonging to the transferrin family, produced by epithelial cells, which is very common in secretory fluids such as milk, saliva, tears or gastrointestinal secretion (14). It is also one of the main components in the granules of neutrophils, from which it is released during inflammation (15), therefore an increased level of this protein is considered by some authors to be an important indicator of inflammation (9, 15). Salivary lactoferrin is produced in the epithelial cells of the serous salivary glands. It may be also derived from plasma penetrating into saliva during inflammation of the mucosa and/or salivary glands (6) or form as a product of degradation of granulocytes located in the gingival sulcus (4). Lactoferrin has biostatic and biocidal effects against a number of bacteria (including S. mutans, S. mitis, S. salivarius), fungi (C. albicans) and viruses (HPV1) (9, 16). The antimicrobial activity of lactoferrin is related to e.g. its high capacity of binding iron ions, and thus depriving pathogens of this element (13, 17). Additionally, when degraded by pepsin, lactoferrin releases peptides showing direct bacteriostatic and antifungal activity (12). Lactoferrin also exerts immunomodulating effects, e.g. by stimulating lymphocytes to increase TNF-α and INF-γ cytokine production as well as by stimulating neutrophil phagocytosis and the release of interleukin (Il)-8 (18). Furthermore, lactoferrin regulates the production of GM-CSF (granulocyte-macrophage colony-stimulating factor) in macrophages (14) and impairs microbial adhesion to tissues, including the adhesion of Streptococcus mutans to tooth enamel hydroxyapatite (18).
Aim
The aim of the study was to analyse the levels of selected non-specific immune components of mixed saliva, i.e. lysozyme and lactoferrin, in patients in the late period (i.e. more than 100 days) after allogeneic hematopoietic cell transplantation. It was also evaluated whether there were significant differences in mixed saliva levels of lysozyme and lactoferrin, depending on the time elapsed since transplantation. Furthermore, the relationship between mixed saliva levels of lysozyme and lactoferrin and the salivary flow rate was assessed.
Material and methods
A total of 45 patients (17 women and 28 men) 3.5 months up to 5 years after allogeneic HSCT, remaining under the care of the Department of Haematology and Transplantation at the Medical University of Gdańsk and the Department of Conservative Dentistry at the Medical University of Gdańsk were included in the study. The study was approved by the Bioethics Committee of the Medical University of Gdańsk (No. NKEBN/886/2004). All tests were performed in accordance with the recommendations of the Helsinki Convention.
Patients were divided into three groups, depending on the time elapsed since transplantation, to include the gradual process of hematopoietic reconstitution after allogeneic HSTC in the assessment. Group I included patients 3.5 to 10 months after transplantation (i.e. in the phase of significant hematopoietic immaturity). Group II included patients 12 to 24 months after transplantation (i.e. in the phase of progressive stabilization of the hematopoietic system). Group III included patients more than 24 months after transplantation (i.e. in the phase of full hematopoietic maturity). Control group included 27 healthy individuals who consented to participate in the study (tab. 1).
Tab. 1. Patient characteristics, taking into account the time elapsed since allogeneic HSCT
| Group | Number of subjects | Time since allo-HSCT (months) | Age (years) |
| n | ? ± δ | Me | range | ? ± δ | Me | range |
| I | 20 | 5.9 ± 2.3 | 6 | 3.5-10 | 41.6 ± 10.4 | 46 | 22-54 |
| II | 14 | 19.1 ± 3.2 | 18.5 | 12-24 | 31.4 ± 7.7 | 30 | 21-46 |
| III | 11 | 36.9 ± 10.8 | 36 | 27-66 | 40.4 ± 10.8 | 40 | 19-54 |
| In total | 45 | 17.6 ± 13.7 | 17 | 3.5-66 | 38.1 ± 10.1 | 38 | 19-54 |
Stimulated and resting mixed saliva was taken from all patients before noon (the patients refrained from eating, tooth brushing, smoking, chewing gum, and also drinking, unless precluded by their general condition or the degree of mouth dryness, for two hours before taking samples). Collection of resting saliva involved 2-minute salivary accumulation in the mouth followed by spitting into a calibrated test tube of a Corning type. This activity was repeated three times, which gave the total time of saliva collection equal to 6 minutes. The total volume of the collected resting saliva was divided by 6 to obtain the amount of saliva secreted during one minute (ml/min). Salivary stimulation involved chewing a paraffin block for 6 minutes. The first portion of saliva (after 1-minute stimulation) was swallowed by the patient, while the subsequent portions, secreted during the remaining 5 minutes of chewing, were collected in the test tube. Next, the amount secreted during one minute (ml/min) was calculated based on the total volume of the collected stimulated saliva. Lactoferrin and lysozyme levels were assayed in the centrifuged mixed resting and stimulated saliva (10,000 x g; 10 min). Biochemical salivary testing was performed in the laboratory of the Department of Conservative Dentistry at the Medical University of Gdańsk. A two-step method based on ELISA was used for lysozyme and lactoferrin level assessment (19). Human milk lysozyme (Sigma-Aldrich) was used as a reference. The obtained results are given in μg/ml. The non-parametric Mann-Whitney U test and Spearman’ rank correlation were used in statistical analysis. The level of significance was p < 0.05.
Results
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